The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Because of their excellent metal chelating properties derivatives of azamacrocycles are widely used as organic ligands in magnetic resonance imaging (MRI) and positron emission tomography (PET) [Aime, S., Botta, M., Fasano, M. and Terreno, E. 1998, Chem. Soc. Rev., 27, 19, Caravan, P., Ellison, J. J., McMurry, T. J. and Lauffer, R. B., 1999, Chem. Rev., 99, 2293, Woods, M., Kovacs, Z. and Sherry, A. D., 2002, J. Supramol. Chem., 2, 1]. Covalently linked to bioactive molecules certain azamacrocycles have been used as target-specific radiopharmaceuticals [Volkert, W. A. and Hoffman, T. J., 1999, Chem. Rev. 99, 2269, Liu, S., Scott, D. S., 2001, Bioconjugate Chem., 12, 7]. Oligonucleotides tethered to triazacycloalkanes, in turn, have been shown to be site-specific RNA cleavage agents [Niittymäki, T. Lönnberg, H., 2006, Org. Biomol. Chem. 4, 15] which may be used in the future in chemotherapy as chemically reactive antisense oligonucleotides. Lanthanide(III) chelates based on 1,4,7-triazacyclononane are among the brightest stable lanthanide(III) chelates synthesized [Takalo, H., Hemmilä, I., Sutela, T., Latva, M., 1996, helv. Chim. Acta, 79, 789, Ziessel, R., Charbonniere, L. J., 2004, J. Alloys Comp. 374, 283]. These types of labels may be extremely useful in in vitro diagnostics based on time-resolved fluorescence technology.
In several applications, covalent conjugation of azamacrocycles to bioactive molecules is required. Most commonly, this is performed in solution by allowing an amino or mercapto group of a bioactive molecule to react with isothiocyanato, maleimido or N-hydroxysuccinimido derivatives of the azamacrocycle [Fichna, J., Janecka, A., 2003, Bioconjugate Chem., 14, 3]. Since in all the cases the reaction is performed with an excess of an activated label, laborious purification procedures cannot be avoided. Especially, when attachment of several label molecules, or site-specific labeling in the presence of several functional groups of similar reactivities is required, the isolation and characterization of the desired biomolecule conjugate is extremely difficult, and often practically impossible.
For oligonucleotide derivatization, an alternative tethering strategy has been developed. It involves solid phase synthesis of oligonucleotides containing an electrophilic ester [Hovinen, J., Guzaev, A., Azhayev, A., Lönnberg, H., 1994, Tetrahedron, 50, 7203, Hovinen, J., Guzaev, A., Azhayev, A., Lönnberg, H., 1994, J. Chem. Soc. Perkin Trans 1, 2745] or thioester linker [Hovinen, J., Guzaev, A., Azhayeva, E., Azhayev, A., Lönnberg, H., 1995, J. Org. Chem. 60, 2205, Hovinen, J. Salo, H., 1997, J. Chem. Soc. Perkin Trans 1, 3017], and cleavage of the linker with appropriate azamacrocycles tethered to an amino group [Hovinen, J. 1998, Bioconjugate Chem. 9, 132] giving rise to oligonucleotide conjugates where the azamacrocycle is incorporated to the oligonucleotide structure via an amide bond. Similar tethering strategy has been applied also to oligopeptide derivatization [Tuchscherer, G., 1993, Tetrahedron Lett., 34, 8419]. Although the derivatization proceeds smoothly and the desired biomolecule conjugates can be obtained in high yield, a large excess of the nucleophile, i.e. the azamacrocycle is required. Accordingly, the method is not practical if complicated structures have to be incorporated. In these cases it is highly desirable to couple the label to the biomolecule structure during chain assembly as appropriate building blocks. Accordingly, the purification problems can be avoided by performing the labeling reaction on solid phase. Hence, most of the impurities can be removed by washings when the biomolecule conjugate is still anchored to the solid support, and after release to the solution, only one chromatographic purification is needed. Synthesis of such blocks which allow introduction azamacrocycles to oligopeptides [Heppeler, A., Froilevaux, S., Mäcke, H. R., Jermann, H. E., Béhé, M., Powell, P., and Hennig, M., 1999, Chem. Eur. J., 1, 1974, De Leon-Rodriguez, Kovacs, Z., Dieckmann, G. R., Sherry, A. D., 2004, Chem. Eur. J., 1, 1974, 10, 1149.] and oligonucleotides have been demonstrated.
Solution phase labeling of large biomolecules, such as proteins cannot be avoided. In these cases, the labeling reaction has to be as selective, and purification of the biomolecule conjugate as effective as possible.
Although numerous excellent MRI contrast agents have been synthesized [Raymond, K. N., Pierre, V. C., 2005, Bioconjugate Chem., 16, 3], the macrocyclic chelator, Gd(III) DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, Dotarem) is still one of the most commonly used. However, the properties of Gd(III) DOTA are not optimal. The net charge of −1 may cause problems in several applications. The chelate distributes thorough the extracellular and intravascular fluid spaces, but does not cross an intact blood-brain barrier. Naturally, bioactive molecules labeled with this type of chelates have lower cell permeability than the corresponding intact molecules [Rogers, B. E., Anderson, C. J., Connett, J. M., Guo, L. W., Edwards, W. B., Sherman, E. L., Zinn, K. R., Welch, M. J., 1996, Bioconjugate Chem. 7, 511]. Furthermore, the negatively charged chelates may bind unselectively to positively charged binding sites of target molecules, such as antibodies, via electrostatic interactions which may result in low recoveries [Rosendale, B. E., Jarrett, D. B., 1985, Clin. Chem., 31, 1965]. Naturally, all these above mentioned problems will be even more serious when the target molecule is labeled with several charged chelates [Peuralahti, J., Suonpää, K., Blomberg, K., Mukkala, V.-M., Hovinen, J. 2004, Bioconjugate Chem. 15, 927]. To overcome these problems, several neutral chelating agents have been synthesized, among which chelates based on 1,4,7-triazacyclononanetriacetic acid (TETA) are the most common. Although the water proton relaxivity of Gd(III) (TETA) is higher than that of Gd(DOTA), its kinetic and thermodynamic stabilities are lower. Accordingly, Gd(III) chelates based on 1,4,7-triazacyclodecane (DETA) are the compromise of neutrality, high water relaxivity and stability [Bucher, E., Cortes, S., Chavez, F., Sherry, A. D., 1991, Inorg. Chem., 30, 2092]. Introduction of a substitutent at one of the carbon on of a substitutent at one of the carbon atoms further enhances these properties.
One of the challenges on the preparation of azamacrocyles has been the cyclization reaction, since addition of the linker required for biomolecule derivatization to one of the reactants often decreases the yield of the desired macrocycle dramatically. Recently, the problem has been solved by performing the cyclization reaction between an appropriately substituted diol and a pernosylated amine under Mitsunobu conditions [Hovinen, J., Sillanpää, R. 2005, Tetrahedron Lett., 46, 4387]. In this reaction bulky substituents in the close proximity of the reaction centres facilitate the formation of the azamacrocycle.
An additional challenge on the preparation of azamacrocycles is their chemical nature. Since the unprotected azamacrocycles are basic, highly polar, nonchromophoric compounds, their purification and isolation is difficult.